This invention relates to an improved test for diabetes mellitus ("diabetes") that can be used even with a patient having levels of substances that would otherwise interfere with testing, e.g., elevated (high) uric acid/and or bilirubin levels. Broadly speaking, this invention concerns a method of testing that treats a specimen from an individual to substantially reduce or eliminate interfering substances from the specimen, assays a material derived from the treated specimen for a certain clinical value (protein-bound glucose level), obtains a second clinical variable for that individual (glucose level), and then uses those two clinical values to assess the likelihood of that individual having diabetes, e.g., by placing the individual in any one of several categories, which categories are associated with pre-established risks of having diabetes.
As is well known, diabetes is a serious disease affecting a significant portion of the population. Detecting whether an individual has diabetes and monitoring diabetes therapy are some of the problems confronting medicine. An early screening test for diabetes and one that is still commonly used involves determining an individual's blood glucose level. See, e.g., U.S. Pat. Nos. 2,981,606; 3,653,841; 3,791,988; and 3,920,580 (all of the patent and other documents, including literature articles, cited or otherwise identified in this application are hereby incorporated herein in their entireties for all purposes). Glycosylated amino acids in urine have also been used to screen for diabetes. See U.S. Pat. No. 4,371,374. U.S. Pat. No. 4,397,956 concerns a single-reading pseudo-kinetic method for monitoring the status of control of ketoacidosis-prone diabetics by measuring the blood glucose and at least one additional indicator analyte (e.g., ketone bodies such as acetone, beta-hydroxybutyrate, and acetoacetate and fatty acid derivatives).
One problem with glucose tests is that even in an individual who does not have diabetes, his or her glucose level can vary over a wide range, depending on when the test sample or specimen (e.g., blood) was taken and when and what the individual last ate. Furthermore, even if one glucose test gives a high enough reading to strongly suggest the presence of diabetes, that individual must undergo additional tests, for example, a so-called glucose tolerance test, before a diagnosis of diabetes can be confirmed. The glucose level of a diabetic taking insulin can also vary dramatically depending upon when the individual last took insulin and on the dosage. It is not unknown for individuals who have diabetes to take insulin shortly before specimens are taken from them for therapy-monitoring glucose tests, so that their glucose tests will indicate normal levels of glucose and make it appear that the individuals have been conscientiously following their prescribed regimens of insulin therapy. That makes monitoring such therapy more difficult. For all these reasons, glucose testing alone was and is known to have significant disadvantages.
Reactions of sugars and amino compounds (for example, proteins) to form N-substituted glycosylamines, which undergo subsequent irreversible Amadori rearrangement, have been known for decades. See, e.g., Hodge, Agricultural And Food Chemistry, vol. 1, no. 15, pp. 928-943 (Oct. 14, 1953). Hodge also noted at p. 930 that investigators showed that glucose and the free amino groups of bovine serum albumin and other proteins and peptides combined in a one-to-one molar ratio.
Years ago it was discovered that in a human diabetic, hemoglobin reacts with glucose in the blood to produce glycated (glycosylated) hemoglobin and that the level of glycated hemoglobin can be determined and used to detect diabetes and to monitor the course of therapy. Hemoglobin is the most abundant protein found in whole blood and its half-life is about 60 days. Glycated hemoglobin forms when glucose binds to the amino moieties of the hemoglobin. The bound glucose moiety undergoes Amadori rearrangement to form a fructose moiety. Both glucose and fructose are reducing sugars, that is, they can reduce other compounds (donate electrons to the other compounds) under the appropriate reaction conditions. Because of the relatively lengthy half-life of hemoglobin, glycated hemoglobin is insensitive to short-term variations in glucose levels, such as might be caused by taking a large dose of insulin or eating sugar-containing candy. Thus, it was discovered that the glycated hemoglobin level indicated an individual's long-term blood glucose history. See, e.g., U.S. Pat. Nos. 4,200,435; 4,243,534; 4,260,516; 4,268,270; 4,269,605; 4,399,227; 4,407,961; and 4,409,335.
There are other proteins in blood besides hemoglobin, and years ago it was discovered that in a diabetic those other proteins also become glycated to a greater or lesser degree. Those proteins are reported to have half-lives of anywhere from 2.5 to 23 days. In particular, the half-life of albumin is reported to be 14 to 20 days and will be taken as 19 days for purposes of further discussion. Albumin (at about 35-50 grams per liter) and globulins (at about 20-30 grams per liter) are the principal proteins in serum. (Removal of blood cells from whole blood yields plasma, and coagulation of the fibrinogen in the plasma and removal of the resulting fibrin yields serum.) When serum proteins are glycated in vivo, glycated albumin usually accounts for about 80 percent of the glycated serum proteins (that is, protein-bound glucose).
Accordingly, detection of serum glycated proteins (sometimes called "fructosamines" in the literature) became another suggested method for detecting diabetes and monitoring its therapy. See, for example, U.S. Pat. Nos. 4,642,295; 4,645,742; 4,797,473; 4,956,301; 5,055,388; Japan Patent Application No. 63-180861; Schleicher et al., J. Clin. Chem. Clin. Biochem., vol. 19, pp. 81-87 (1981); Dolhofer et al., Clinica Chimica Acta, vol. 112, pp. 197-204 (1981); Johnson et al., Clinica Chimica Acta, vol. 127, pp. 87-95 (1982); Armbruster, Clin. Chem., vol. 33, no. 12, pp. 2153-2163 (1987); Rosenthal et al., Clin. Chem., vol. 34, no. 2, pp. 360-363 (1988); Caines et al., Clin. Biochem., vol. 19, pp. 26-30 (February. 1986); Jue et al., J. Biochem. Biosphys. Meth., vol. 11, pp. 109-115 (1985); Armbruster, Clin. Lab. Sci., vol. 3, no. 3, pp. 184-188 (May/June 1990); and Windeler et al., J. Clin. Chem. Clin. Biochem., vol. 28, pp. 129-138 (1990).
Various methods have been used to detect fructosamine, which is referred to herein as "protein-bound glucose" or "PBG." Use of the word "fructosamine" to refer to a protein-glucose Amadori rearrangement product is undesirable in part because it is not a recognized American Chemical Society or Chemical Abstracts protein chemical name or category and because it is also the trivial chemical name of a relatively simple compound unrelated to proteins or protein-sugar adducts. Hence the term "protein-bound glucose," which is more accurate and whose meaning is clear, is preferred and is used herein. Current procedures for measuring serum glycated proteins (i.e., protein-bound glucose) include affinity chromatography, agarose gel electrophoresis, high-performance liquid chromatography (HPLC), immunoassay with monoclonal or polyclonal antibodies, and colorimetric methods.
It was known that under the appropriate conditions, typically alkaline pH, certain compounds that were otherwise colorless in solution would be reduced by (that is, receive electrons from) the reducing sugar moiety of the protein-bound glucose and become colored. It was also known that the reaction conditions could be chosen so that the intensity of the color would be directly proportional to the concentration of protein-bound glucose in the sample being tested. For example, tetrazolium and other compounds have been used as color indicators. See, e.g., U.S. Pat. Nos. 3,576,815; 3,791,988; 4,642,295; 4,645,742; 4,956,301; Mattson et al., Anal. Chem., vol. 22, no. 1, pp. 182-185 (January. 1950); Mopper et al., Anal. Biochem., vol. 45, pp. 147-153 (1972); Chem. Abstr., vol. 95, entry 148753e (Foods, 1981); Caines, Clin. Biochem., vol. 19, pp. 26-30 (February 1986); Jue et al., J. Biochem. Biophys. Meth., vol. 11, pp. 109-115 (1985); and Chem. Abstr., vol. 82, entry 70120f (Biochem. Meth., 1975). An assay marketed by Roche Diagnostic Systems, Inc., Montclair, N.J., uses nitroblue tetrazolium ("NBT"). An assay marketed by Isolab Inc., Akron, Ohio, uses 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ("INT"). Trudinger, Anal. Biochem., vol. 36, pp. 222-224 (1970), reports that methyl viologen is reduced by alkaline glucose to form a colored solution. Other compounds are known (including benzyl viologen) that change color and color intensity under the appropriate reaction conditions in the presence of reducing sugars or moieties of reducing sugars attached to proteins.
Johnson et al., Clinica Chimica Acta, vol. 127, pp. 87-95 (1982), report the advantages of measuring protein-bound glucose as an index of diabetic control. However, one recent review article concludes, based upon an analysis of literature articles concerning this type of test, that the test has not been evaluated sufficiently to allow its routine clinical use and that the results reported for it in the literature do not suggest that the test is reliable. See Windeler et al., J. Clin. Chem. Clin. Biochem., vol. 28, pp. 129-138 (1990). The manufacturer of one commercial assay recently referred to the growing unease with the fructosamine assay, said that people are beginning to feel that the method has not lived up to its promise, and noted three technical problems with it. Isolines, vol. 19, no. 3, p. 1 (Isolab Inc., October 1990).
The literature also reports that various substances can interfere with assays (and particularly colorimetric assays) for protein-bound glucose, e.g., non-specific binding agents and, if elevated, uric acid and bilirubin. Interference is one of the three problems noted by Isolab (Id.). Elevated uric acid and/or bilirubin can be the result of disease or other process (e.g., liver disease, cancer). Approximately 1% of the general population and 6-7% of the hospital population have elevated levels. Colorimetric tests for protein-bound glucose in individuals having sufficiently elevated bilirubin and/or uric acid levels are viewed as unreliable and such tests are generally not run for those individuals.
U.S. Pat. Nos. 4,642,295 and 4,645,742, which use the coloring agent nitroblue tetrazolium ("NBT") under alkaline conditions to indicate the presence and concentration of protein-bound glucose ("PBG"), require that two colorimetric intensity readings be taken to try to reduce the adverse effects of certain interference. The first reading is taken after a suitable delay from the addition of NBT to the specimen (e.g., 10 minutes) to allow non-PBG/tetrazolium reactions to occur and the second reading is taken after a suitable delay following the first reading. If the first intensity reading is taken before substantially all of those non-specific (i.e., non-PBG) reactions have occurred, the difference in intensities between the two readings will include the intensity change caused by reaction of non-PBG compounds with the tetrazolium and that may introduce a significant error into the calculated PBG level. Because the NBT method uses two readings at different times following the addition of NBT to the specimen, it is also referred to as "the kinetic method." However, this method does not eliminate the adverse effects caused by elevated levels of uric acid and/or bilirubin.
It is known to add polyethylene glycol to serum to precipitate both glycated and non-glycated globulins while leaving glycated and non-glycated albumin and smaller molecules (e.g., uric acid and bilirubin) in solution, i.e., in the supernatant, and then to test the solution for glycated albumin by the nitroblue tetrazolium colorimetric method. That separation (precipitation) method leaves the non-protein, low molecular weight interfering substances in solution with the albumin, possibly to interfere with the colorimetric test if their levels are high enough. It is also known to use affinity chromatography on serum to separate glycated serum proteins (principally glycated albumin and glycated globulins) from the other substances present in the serum. These other substances do not bind to the column and include non-glycated proteins and the non-protein, low molecular weight interfering substances. The glycated proteins bound in the column are eluted and the protein concentration is measured by colorimetric or non-colorimetric methods. Mashiba et al., "Measurement of Glycated Albumin by the Nitroblue Tetrazolium Colorimetric Method," Clinica Chimica Acta, vol. 212, pp. 3-15 (1992). See also Armbruster, Clin. Chem., vol. 33, no. 12, pp. 2153-2163 (1987). It is also known to add uricase to serum (to eliminate uric acid) along with the colorimetric reagent and then colorimetrically measure the glycated proteins in the serum.
Researchers disagree as to the effects of albumin on the NBT (nitroblue tetrazolium) test. Johnson et al., Clinica Chimica Acta, vol. 127, pp. 87-95 (1982), indicate that in the NBT test, correcting fructosamine for albumin concentration makes the difference between normal and diabetic sera less clear. On the other hand, Armbruster, Clin. Chem., vol. 33, no. 12, pp. 2153-2163 (1987), a survey article, reports at pp. 2157-2158 conflicting opinions from other researchers. Some researchers said that hypoalbuminemia (clinically significant low albumin level) influenced the NBT assay only when the albumin concentration was less than 30-35 grams/liter. Other researchers found that protein-bound glucose values from the NBT method were affected by albumin regardless of the albumin concentration and they suggested a certain correction factor based on albumin concentration. Other researchers recommended a different correction based on albumin concentration. Yet other researchers did not find fructosamine to be significantly influenced by albumin concentration.
Rosenthal et al., Clin. Chem., vol. 34, no. 2, pp. 360-363 (1988), report on a single-color-reading method for determining protein-bound glucose. At p. 362 they surmise that fructosamine concentrations from their method are affected by serum albumin concentrations in the same manner as the two-color-reading or kinetic method and that this should be taken into account for patients whose serum albumin concentrations are abnormal. Finally, Armbruster, Clin. Lab. Sci., vol. 3, no. 3, pp. 184-188 (May/June 1990), another survey article, reports at p. 187 that the drawbacks of the NBT method include the nonspecific nature of the reaction in serum, the effects of albumin concentration, and how to best calibrate the test. Armbruster recommends that determining glycated hemoglobin instead of determining protein-bound glucose may be more advantageous when a patient's albumin or total protein values are low (Id.).
Thus, there are conflicting opinions in the literature as to whether detecting protein-bound glucose in serum is a reliable and accurate way to test for diabetes or to monitor the course of therapy, whether the presence of albumin significantly affects the NBT test for protein-bound glucose and if so, how to compensate for that effect, and whether an assay for determining protein-bound glucose should be used at all for patients who have low albumin levels.
Furthermore, no reliable method exists for eliminating or significantly reducing the adverse effects of, e.g., elevated uric acid and/or bilirubin levels in PBG assays, particularly colorimetric assays. Accordingly, there is a continuing need for a reliable, reproducible, relatively rapid, and relatively inexpensive method for testing individuals for diabetes with high accuracy and for monitoring the course of diabetes therapy, even if those individuals have levels of uric acid, bilirubin, and/or other substances that would otherwise interfere with determination of their protein-bound glucose.